Acute effects of 17ß-estradiol on myocardial pH, Na, and Ca and ischemia/reperfusion
Steven E. Anderson, Dawn M. Kirkland, Andrea Beyschau, and Peter M. Cala
Department of Human Physiology
University of California
Short title: Estrogen, H, Na, and Ca in myocardial I/R injury
Subject codes: Ischemic biology - basic studies, Ion channels/membrane transport,
Address all correspondence to:
Department of Physiology and Membrane Biology
University of California
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Articles in PresS. Am J Physiol Cell Physiol (September 22, 2004). doi:10.1152/ajpcell.00414.2004
Copyright © 2004 by the American Physiological Society.
ABSTRACT Evidence suggests ischemia/reperfusion injury is largely due to cytosolic Ca
accumulation resulting from functional coupling of Na/Ca exchange (NCX) with stimulated Na/H
exchange (NHE1). Evidence also suggests 17ß-estradiol (E2) stimulates release of NO which
inhibits NHE1. Thus we tested the hypothesis that acute E2 limits myocardial Na and therefore
Ca accumulation and thereby limits ischemia/reperfusion injury. NMR measured cytosolic pH
(pHi), Na (Nai) and calcium concentration ([Ca]i) in Krebs-Henseliet-perfused hearts from
ovariectomized rats (OVX). Left ventricular developed pressure (LVDP) and lactate
dehydrogenase (LDH) release were also measured. Control ischemia/reperfusion was 20 min of
baseline perfusion, 40 min global ischemia, and 40 min of reperfusion. The E2 protocol was
identical except 1 nM 17ß-estradiol was included in the perfusate prior to ischemia and during
reperfusion. Data are expressed as mean±SEM and the following differences were found,
p<0.05. E2 limited the changes in pHi, Nai, and [Ca]i during ischemia. For control OVX vs.
OVX+E2: pHi fell from 6.93±0.03 to 5.98±0.04 vs. 6.96±0.04 to 6.68±0.07; Nai (mEq/kg dry
wt) rose from 25±6 to 109±14 vs. 25±1 to 76±3; [Ca]i (nM) changed from 365±69 to 1248±180
vs. 293±66 to 202±64. E2 also improved recovery of LVDP and diminished release of LDH
during reperfusion. Effects of E2 were diminished by 1 µM Nω-nitro-L-arginine methyl ester
(LNAME). Thus, the data are consistent with the hypothesis. However, E2 limitation of
increases in [Ca]i is greater than can be accounted for by the thermodynamic effect of reduced Nai
accumulation on NCX.
Keywords: myocardial ischemia, Na/H exchange, Na/Ca exchange, 17ß-estradiol, NMR
It is clear that our understanding of the effects of endogenous and exogenous estrogen on
the cardiovascular system and in particular on its role in modifying susceptibility to ischemic
injury is deficient. It is more important than ever that fundamental research be conducted to
understand the basis for the effects of estrogen (50). Because results of various studies are
inconsistent and/or difficult to interpret we have taken a reductionist approach to testing the
acute effects of exogenous 17ß-estradiol (E2) on ischemic myocardium within the context of the
well accepted paradigm that ischemic injury is largely the result of the following chain of events.
Anaerobic metabolism decreases cytosolic pH (pHi) which stimulates pH-regulatory Na/H
exchange (NHE1) to increase Na uptake and thereby increase intracellular Na (Nai). Increases in
Nai decrease the driving force for cytosolic Ca efflux via Na/Ca exchange (NCX) and thereby
cause an increase in cytosolic [Ca] ([Ca]i). Increases in [Ca]i cause a cascade of events ending in
injury, apoptosis, or necrosis (3, 7, 31, 37, 46, 49). In the heart, there are questions about
whether the bulk of Na and Ca entry is during ischemia or reperfusion, but there is a growing
consensus that much of the Ca entry is Na dependent (36). Numerous studies show E2
stimulates NOS activity and/or release of NO in the heart (20, 39, 40) and that female hormone-
linked changes in Na and Ca accumulation and protection of myocardium from
ischemia/reperfusion injury is NO dependent (14-16). Although these studies implicate the
sarcolemmal Na/Ca exchanger (NCX) in the gender dependent response they do not make it clear
whether the effect of female hormones is directly on NCX itself or on other transporters which
contribute to changes in the Na or Ca gradients or Em, the thermodynamic determinants of flux
via NCX (38, 46). Studies conducted on isolated mouse cardiac myocytes overexpressing NCX
and using a metabolic inhibition protocol to mimic ischemia argue Nai-dependent increases in
[Ca]i are diminished by female hormone but the mechanism of Nai accumulation was not
determined (48). More recent experiments from the same laboratory concluded the acute
cardioprotective effect of E2 during metabolic inhibition may be mediated by an anti-oxidant
effect (47). The latter studies, however, were conducted using a dose of 100 nM E2. This dose
is nearly 100 times greater than peak E2 in cycling human females and nearly 500 times greater
than that in mice. Thus the mechanism of the E2 effect(s) on cytosolic Na and Ca and protection
from ischemic injury for physiological levels of E2 remains unclear.
Given aforementioned evidence that E2 stimulates release of NO and NO inhibits NHE1
in cardiac myocytes (22), we tested the hypothesis that acute exposure of isolated rat hearts to
E2 will limit Na uptake during ischemia/reperfusion and thus limit Na-dependent increases in
[Ca]i as well as associated ischemia/reperfusion injury. The hypothesis further predicts this
effect of E2 will be inhibited by the NO synthesis inhibitor LNAME.
Additionally, in order to more directly test the hypothesis that E2 inhibits NHE1, we
used the NH4Cl washout protocol (8) to acidify the heart under normoxic, normal perfusion,
HEPES buffered conditions ± E2. This protocol allows one to assess the response of NHE1 to
intracellular acidification similar in magnitude to that which occurs during ischemia but with fewer
uncontrolled parameters (28).
The results are consistent with the hypothesis that E2 inhibits NHE1 in an NO-
dependent manner. That is, in the HEPES buffered NH4-washout protocol E2 diminished the
rate of proton efflux. In the ischemia protocol E2 limited cytosolic H, Na, and Ca accumulation
during ischemia. Further, these effects of acute E2 improved recovery of function and limited
LDH release during reperfusion. However, E2 limitation of increases in [Ca]i during ischemia is
greater than can be accounted for by the thermodynamic effect of the Na gradient on NCX,
suggesting E2 also attenuates NCX activity.
Thus the results of these studies provide new insights into the mechanisms of gender- or
female hormone-dependent effects on myocardial ischemia-induced cell Ca accumulation and
injury. They go beyond previous studies to provide strong evidence that acute E2 inhibits NHE1
during ischemia and more generally after intracellular acidification. Furthermore, thermodynamic
arguments provide strong circumstantial evidence that E2 also inhibits NCX independent of
changes in [Na]i during ischemia. While the evidence supports the conclusion that E2 inhibits
NHE1 via NO, it remains to be investigated how it might inhibit NCX. These results have been
previously reported in abstract form (23).
General: The methods used were modified from those previously reported (3, 6, 31).
Briefly, 12-13 week old Sprague-Dawley rats were purchased from Charles River one week after
ovariectomy (OVX). Animals were treated according to Guiding Principles in the Care and Use
of Animals using an IACUC approved protocol. One week after delivery OVX were
anesthetized with sodium pentobarbital (65 mg/kg I. P.) and heparinized (1000 USP units/kg
I.V.). Hearts were removed and the aorta was cannulated and perfused at a constant rate (10-12
ml/min). All experiments were conducted at 36±1°C. Control perfusate contained (mmole/liter)
133 NaCl, 4.75 KCl, 1.25 MgCl2, 1.82 CaCl2, 25 NaHCO3, 11 dextrose, insulin 10 U/liter (44)
and was equilibrated with 95% O2/5% CO2 which provided a pH of 7.35-7.45. Left ventricular
developed pressure (LVDP - end systolic minus end diastolic pressure) was measured by a fluid
filled balloon placed in the left ventricle and secured by means of a ligature immediately proximal
to the mitral valve. Left ventricular pressure and perfusion pressure were monitored
continuously by strain gauge transducers connected to a MacLab. Left ventricular volume was
adjusted as necessary to maintain end-diastolic pressure at 5±5 cm H2O.
The normoxic acidification protocol was conducted using HEPES buffered perfusate (HR)
which was identical to the perfusate described above except 20 mM HEPES acid plus 8 mM
NaOH were substituted for 25 mM NaHCO3 and the perfusate was equilibrated with 100% O2
and titrated to pH 7.40±0.05. The protocol consisted of 20 min baseline perfusion with HR, 35
min perfusion with HR + 10 mM NH4Cl, followed by 30 min perfusion with HR (NH4Cl
washout). For acute E2 and LNAME experiments 1 nM E2 and/or 1 µM LNAME was added
both to the NH4Cl solution (5 min prior to NH4Cl washout) as well as the NH4Cl washout
solution. 31P NMR was used to measure pHi as described below. Proton efflux rates were
calculated as the product of the change in pHi during the first 5 min of recovery after acidification
multiplied by cardiac myocyte buffer capacity. The buffer capacity (mM/pH) equals -28
(pHistart)+222.6 (28) where pHistart is the pHi at the beginning of the recovery interval.
The control ischemia protocol consisted of 30 minutes baseline perfusion followed by 40
minutes ischemia followed by 40 minutes reperfusion. In order to limit glycolysis (1) 5.5 mM 2-
deoxy-D-glucose was substituted for dextrose 10 min prior to ischemia. The E2 protocol was
identical except 1 nM 17ß-estradiol was added to the perfusate used during the baseline and
reperfusion intervals. Each of the protocols was also conducted with the addition of Nω-nitro-L-
arginine methyl ester (1 µM-LNAME) to the perfusate. When LNAME was added it was added
along with E2 or when E2 would have been added. Initiation of 40 min of ischemia was
designated t = 0 min. 23Na, 31P, and 19F NMR were used to measure Nai, pHi and high energy
phosphates, and [Ca]i respectively. In order to measure Nai, 7.5 mmol/L dysprosium
triethylenetetraminehexaacetic acid (DyTTHA) was substituted for NaCl in the perfusate (iso-
osmotically) and Ca was added until the perfusate concentration reached 1.8-2 mmol/L as
measured by Ca electrode (31). In order to measure [Ca]i, hearts were perfused for 30 min with
perfusate containing the acetoxymethyl ester of 5F-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-
tetraacetic acid (FBAPTA) at 8 µmol/L (6). FBAPTA was then washed out of the extracellular
space with control solution for 15 min before baseline measurement of [Ca]i. After perfusions
were complete, hearts used to measure 23Na were weighed wet and dried to constant weight (at
least 48 hr) at 65°C to determine dry weight. Their wet and dry weights were 1.22±.04 and
0.19±0.01 gm, respectively, and there were no significant differences between treatments.
NMR spectroscopy: Experiments were conducted using a Bruker AMX400
spectrometer. 23Na, 31P, and 19F spectra were generated from the summed free induction decays
of 1000, 148, and 1500 excitation pulses (90°, 60° and 45°) using 2K, 4K, and 2K word data files
and ±4000, ±4000, and ±5000 Hz sweep widths, respectively. Data files were collected over 5
min intervals but in order to improve signal-to-noise for 19F measurement of [Ca]i, two five
minute files were added together. Because the NMR signal intensity reflects the average for the
interval over which data were collected, data are represented in the figures as corresponding to the
midpoint of the appropriate 5 or 10 min acquisition interval. NMR data reflect the signal
collected from the entire heart, both ventricles and both atria.
Nai (mEq/kg dry weight) was calculated from the calibrated area of the “unshifted”
intracellular peak after subtracting out the shifted extracellular peak as previously described (3, 5,
31). (Please note: this method measures Nai as an amount and to minimize assumptions we
report it as such.) [Ca]i in nmoles/liter cytosolic water was calculated as the product of the Ca-
FBAPTA dissociation constant (Kd) and the ratio of the areas of the Ca-bound and Ca-free
peaks in the FBAPTA spectrum (6). The Kd for Ca-FBAPTA was 294 nM. Intracellular pH
was determined from the chemical shift of the inorganic phosphate (Pi) resonance [with reference
to control phosphocreatine (PCr)] calibrated at 37°C (2). High energy phosphates are reported
as percent of control peak intensity (6).
To assess ischemia/reperfusion injury, total lactate dehydrogenase (LDH) released was
measured from timed collections of perfusate leaving the heart for 40 min of reperfusion as
previously described for CK (43). LDH in IU/gm dry wt was measured spectrophotometrically
(Pointe Scientific, Lincoln Park, MI).
Unless otherwise stated, results are reported as mean±SEM. For the ischemia
experiments analysis of variance for repeated measures was used to test for differences between
treatments. When differences between treatments were found, the Student-Newman-Keuls
multiple comparison test was used to determine which treatments were different and the times at
which differences between treatments occurred. Only the latter are indicated in figures. For the
NH4Cl washout experiments the Bonferroni correction was used to compare proton efflux rates.
Because overpowered studies are wasteful of resources (17) and/or raise serious ethical issues
when animals are used (30), sample sizes were limited to those required to reject the null
hypotheses with p < 0.05.
Estrogen limits proton efflux via Na/H exchange after normoxic acidification.
Data depicted in Figure 1 demonstrate that acute E2 (1 nM) added to the perfusate 5 min
prior to NH4Cl washout (8) diminishes the proton efflux rate after normoxic acidification in
HEPES buffer (p=0.024) and the effect of acute E2 was diminished by LNAME (p=0.032). In
control OVX the mean proton efflux rate (mM/min) after NH4Cl washout was 1.58±0.23
compared with 0.58±0.22 in OVX+E2 and 1.51±0.30 in OVX+E2+LNAME. There were no
significant differences in pHi between groups after acidification prior to pH regulation (pHstart in
OVX = 6.86±0.06; OVX+E2 = 6.85±0.06; OVX+E2+LNAME = 6.83±0.04. Under these
conditions proton efflux is commonly accepted to occur almost completely via NHE1 (28). Thus
the data support the hypothesis that E2 limits pH-regulatory NHE1 activity. And again, the
observation that addition of 1 µM LNAME significantly limits the E2 effect supports the
hypothesis that the acute E2 effect is mediated by NO.
Estrogen limits intracellular proton accumulation during ischemia.
The data shown in Fig. 2 are consistent with the hypothesis that acute E2 limits the
accumulation of protons in ischemic myocardium (p<0.0001) and that this response is mediated
by NO since it is diminished by 1 µM LNAME (p<0.01). There were no significant differences
between pHi in any of the groups except during ischemia. Under control conditions mean pHi fell
from 6.93±0.03 to 5.98±0.04 during 40 min of ischemia compared with 6.96±0.04 to 6.68±0.07
for OVX+E2, 6.94±0.05 to 6.34±0.13 for OVX+E2+LNAME, and 6.92±0.04 to 6.21±0.08 for
OVX+LNAME. Experiments were also conducted substituting 1 µM DNAME for 1 µM
LNAME in animals exposed to E2. The data are also consistent with the interpretation that
LNAME limits the effect of E2 but DNAME (not shown) does not. That is, in OVX+E2 hearts
pHi was significantly higher during ischemia with DNAME treatment than with LNAME
(p<0.01) and there was no significant difference between OVX+E2 hearts with and without
DNAME. The fact that LNAME alone (closed circles) limited the ischemia-induced fall in pHi
suggests there is a NO-dependent response to ischemia which tends to lower pHi in the absence
of E2. We cannot explain this response, but it does not alter the conclusion that the acute effect
of E2 on pHi during ischemia is diminished by LNAME and thus must be NO-dependent.
Estrogen limits intracellular sodium accumulation during ischemia.
The data in Fig 3 are consistent with the hypothesis that E2 inhibits intracellular Na
accumulation during ischemia (p<0.05) and that this response is mediated by NO since it is
diminished by 1 µM LNAME (p<0.01). Under control conditions mean Nai (mEq/kg dry wt)
rose from 25±6 to 109±14 during 40 min of ischemia compared with 25±1 to 76±3 for OVX+E2,
34±10 to 136±6 for OVX+E2+LNAME, and 31±5 to 129±9 for OVX+LNAME. There were no
significant differences between any groups before or after ischemia except that Nai in the
OVX+E2+LNAME group was greater than in the OVX+E2 group for the first interval of
reperfusion (132±18 vs 52±5; p<0.05).
Estrogen limits increases in intracellular [Ca] during ischemia.
The data in Figure 4 demonstrate that E2 limits cytosolic Ca accumulation during ischemia
(p<0.01) and this response is inhibited by 1 µM LNAME (OVX+E2+LNAME vs. OVX+E2 -
p<0.05). Mean cytosolic [Ca] (nM) rose during 40 min of ischemia from 365±69 to 1248±180 in
control OVX compared with 293±66 to 202±64 in OVX+E2, 232±54 to 900±154 in
OVX+E2+LNAME, and 486±126 to 1700±291 in OVX+LNAME. This result is consistent
with the hypothesis that an NO-dependent effect of E2 inhibits cytosolic Ca accumulation during
Estrogen limits LDH release and improves recovery of function after ischemia
The data are also consistent with the hypothesis that E2, by diminishing cytosolic Ca
accumulation during ischemia, improves recovery of LVDP during reperfusion (p<0.05 - Figure
5) and limits injury as indicated by the decrease in LDH release during reperfusion (p<0.05 -
Figure 6). Under control conditions mean LVDP (% of baseline) rose to 2±1 after 40 min
reperfusion compared with 19±6 for OVX+E2, 4±2 for OVX+E2+LNAME, and 10±5 for
OVX+LNAME. There were no significant differences in LVDP between any groups before or
during ischemia. Again, as predicted by the hypotheses, LDH release during reperfusion was
diminished by acute E2 (p<0.05). During reperfusion LDH release (IU/gm dry wt) in the control
OVX group was 94±43 compared to 18±7, 52±17, and 113±26 in the OVX+E2,
OVX+E2+LNAME, and OVX+LNAME groups, respectively.
The general hypothesis that ischemia/reperfusion (IR) injury is largely the result of
cytosolic Ca accumulation mediated by Na/Ca exchange (NCX) subsequent to pH-regulatory,
Na/H exchange (NHE1) mediated increases in Nai is widely accepted (7, 31, 37, 45). It has also
been well demonstrated that 17ß-estradiol (E2) increases activity of NOS and NO release during
ischemia and reperfusion (20, 39, 41). Furthermore, there is good evidence that the NO donor
sodium nitroprusside as well as 8Br-cGMP inhibit pHi recovery in cardiac myocytes from male
rats after NH4Cl washout in HEPES medium (22). The latter supports the conclusion that NO
(via cGMP) inhibits pH-regulatory Na/H exchange in the heart. Given these findings, we tested
the logical, but previously unarticulated, hypothesis that acute exposure to E2 prior to
acidification would cause an NO-dependent limitation of NHE1. In the context of the HEPES
buffered NH4Cl protocol this would cause an LNAME-inhibitable decrease in proton efflux. In
the context of the ischemia protocol this would cause and LNAME inhibitable decrease in
cytosolic Na and Ca accumulation. The general hypothesis led us to further postulate that E2-
dependent limitation of increases in [Ca]i would limit ischemia/reperfusion-induced myocardial
injury. The data presented here are all consistent with these hypotheses and thus provide new
insight into the mechanism(s) of estrogen's effects on myocardial Nai, Cai, and injury during
Because the balance of proton production and efflux is so difficult to predict and control
during ischemia it is very hard to unequivocally determine the cause and effect relationships
between changes in pHi and [Na]i using that model. Therefore, we tested the hypothesis that E2
will inhibit NHE1 under normoxic, normal flow conditions of intracellular acidification using the
NH4Cl prepulse technique under HEPES buffered conditions (8, 22). Under these conditions a
known amount or bolus of protons can be "injected" into the cell and the rate of proton efflux
during pHi recovery can be ascribed to flux via NHE1 (28). The results of these experiments
provide strong evidence that E2 inhibits NHE1 (Fig. 1) and since the effect of E2 is diminished
by LNAME this result is consistent with the postulate that the E2 effect is NO mediated.
Further consistent with the hypotheses, acute E2 limits cytosolic Na and Ca
accumulation during ischemia and again the effect of E2 is diminished by LNAME indicating it is
mediated by NO (Figs 3 & 4).
The observation that E2 limits the fall in pHi during ischemia might initially seem
contradictory to our hypothesis that E2, via NO, inhibits NHE1. However, we and others have
shown that NHE1 inhibitors either have no effect on, or limit the fall in, pHi during ischemia (31,
42) Furthermore, inhibiting ischemia-induced Na uptake via other pathways also limits ischemia-
induced acidification (9, 24, 34). This is because ischemia induced cytosolic Na and Ca
accumulation stimulate ATP hydrolysis (3, 21, 32) and thus proton production (18). In other
words, during ischemia a positive feedback loop is created whereby increased proton production
increases cytosolic Na and Ca accumulation which stimulates more proton production. If this
occurs, inhibiting accumulation of any of the ions will have a tendency to diminish accumulation
of the others, and in particular limiting increases in [Na]i and [Ca]i will limit proton production
and thereby ischemia-induced acidification (31). The data presented (Fig. 2) fit this paradigm
where acute E2 limitation of cytosolic Na and Ca accumulation (Figs 3 & 4) is associated with a
decreased fall in pHi during ischemia and again, this effect of E2 was diminished by LNAME.
We cannot rule out the possibility that E2 inhibits proton production, e.g. by stimulating ATP-
dependent K channels (29), independent of that due to inhibiting NHE1 (and thus limiting
stimulation of NHE1), but the data in Fig. 1 provide strong evidence that E2 inhibits the response
of NHE1 to acidification under conditions where the acidification is not E2 dependent.
Finally, the finding that acute E2 limits I/R-induced decreases in recovery of LVDP (Fig.
5) and increases in LDH release during reperfusion (Fig. 6) are consistent with the hypothesis
that E2-sensitive Na and Ca accumulation contribute to I/R injury. Although the effects are
statistically significant, in this model LVDP recovery is modest. Thus it remains to be seen
whether the effects of E2 observed in this protocol would be important in a clinical setting.
Although the results of these studies are consistent with the hypothesis that acute E2 will
limit cytosolic Na and thereby Ca accumulation, it should also be noted that with respect to the
data shown in Figs. 3 & 4, the E2-dependent limitation of cytosolic Ca accumulation is greater
than one would expect based upon the reduction in Nai accumulation alone. That is, we and
others have shown that NCX remains near equilibrium under a variety of conditions, and most
notably during ischemia (4, 38, 46). This behavior is interpreted as support for the notion that
increases in [Ca]i during ischemia are mediated by NCX. In other words, if NCX is the dominant
Ca transport pathway the driving force for NCX will be at or near zero; incipient forces will
dissipate due to NCX flux and the pathway will be at equilibrium. If that is the case, the reversal
potential ENCX will equal the cell membrane potential (Em). Conversely, if ENCX is not equal to
Em, NCX is not the dominant Ca transport pathway. If we calculate ENCX at the end of ischemia
after acute E2 (OVX+E2 from the data in Figs 3 & 4) we find ENCX = -126 mV (please see
appendix). Again, this means Em would have to be -126 mV for NCX to be at equilibrium. The
negative limit for Em under essentially all physiological conditions, including those in which K
channels are stimulated, is the potassium equilibrium potential which in this case is near -65 mV
(35). (In fact Em is likely to be nearer -50 mV (46).) Since ENCX is far more negative that Em, (Em
- ENCX ≈ 61 to 76 mV) one may safely argue that after acute E2 treatment NCX is no longer the
dominant Ca transport protein during ischemia.
Thus Occam's razor leads one to conclude that during ischemia after acute E2 NCX must
be inhibited kinetically and/or some other Ca transport pathway is stimulated so that NCX is no
longer dominant. One could argue that there may be errors in the values of [Na] and [Ca] used in
our calculations, but it is unlikely that the errors could be large enough to cause this large
discrepancy between ENCX and Em. First, the error in our assumptions for extracellular [Na] and
[Ca] are likely to be less than 5% and the error in our estimate of Em must be less than 20 mV (Em
cannot be more negative than EK). Second, if the discrepancy were due to errors in measurement
of Nai or Cai it would require an overestimate in our measurement of [Na]i of at least 100% or an
underestimate of [Ca]i by more than 90% to account for the discrepancy. Again, while the above
discussion is based upon formal thermodynamic arguments, the same conclusions can be drawn
qualitatively from the data in Figs. 3 & 4 which show that during ischemia after acute E2
treatment nominal increases in [Na]i are associated with no change or decreases in [Ca]i. This
cannot happen if NCX is the dominant Ca transport pathway.
As mentioned above, one could postulate that NCX is not dominant (at equilibrium)
because E2 stimulates some other pathway to remove Ca from the cytosol, e.g. Ca-ATPase at the
sarcolemma or SR (13). However, in order for the ischemia-induced changes in Nai and Cai to be
dissociated by E2 as observed in Figs 3 & 4, a very large increase in efflux via some other
pathway(s) would be required to surpass flux via NCX unless the latter was inhibited. Thus
again, the simplest explanation for the data includes E2 inhibition of NCX activity. How this
occurs will require further study.
The results of our experiments are consistent with a number of reports which suggest
female hormones play a role in Ca-mediated ischemic injury. These include the studies of Cross
et al. which show that in mutant mice which overexpress either ß2-adrenergic receptors or NCX,
female mice are protected from ischemic injury compared to males and ovariectomized females,
and in the ß2-adrenergic receptor overexpressors, the female resistance to ischemic injury was
reduced by treatment with LNAME (14, 15). In contrast, using a simulated ischemia/reperfusion
protocol with isolated rat astrocytes Matsuda et al. found sodium nitroprusside and 8Br-cGMP
exacerbated injury and the authors concluded NO and cGMP stimulated NCX (33). This is not
consistent with the Cross studies nor ours. On the other hand, in a metabolic inhibition study
using isolated mouse cardiac myocytes overexpressing NCX, Barry and coworkers report
findings consistent with those reported by Cross et al. but include measurement of [Ca]i and
[Na]i consistent with our findings. That is, increases in [Na]i and [Ca]i during metabolic
inhibition were greater in male than female transgenics and gender differences were eliminated by
treating the males with E2 (48). More recent reports from the same group conclude "the acute
cardioprotective effect of estrogen during metabolic inhibition may be mediated by an ER-
independent anti-oxidant action, which results in improved function of Na(+)-K(+) ATPase"
(47). However, the more recent study uses a dose of E2 100 times greater than the dose we use
which raises the question of whether the anti-oxidant effect is physiological.
The fact that the studies we report here were conducted with a 100-fold lower dose of E2
(near peak estrus in the rat) and that the effect is diminished by LNAME (but not DNAME)
provides evidence that the response we observe is not completely due to an anti-oxidant effect of
E2. Furthermore, E2-dependent limitation of increases in intracellular Ca during ischemia seem to
be dependent on the presence of estrogen receptor alpha. Zhai and colleagues report that in
perfused hearts of male mice, estrogen receptor alpha knock outs (ERKO) have increased Ca
accumulation, decreased nitrite production, and increases in multiple indicators of injury during
ischemia/reperfusion (51). Another study from the same laboratory showed that in female OVX
rats chronic E2 treatment was associated with the opposite response to ischemia/reperfusion:
decreased Cai accumulation, increased nitrite production, and decreased injury (52).
Recent studies in a number of organs are consistent with the postulate that NHE1 may be
inhibited by signal transduction pathways associated with increases in NO and specifically the
cascade of steps through NO, cGMP, cGMP-dependent kinase (cGK) and p38 MAPK (19).
These include studies from isolated hepatocytes which suggest a pre-conditioning effect limiting
increases in [Na]i during hypoxia is mediated by NO through cGK and p38 MAPK (11) and
further that atrial natriuretic peptide diminishes hypoxia-induced increases in [Na]i by a similar
pathway which inhibits NHE1 (10). These results are also consistent with studies that suggest
activation of p38 MAPK inhibits the response of NHE1 to angiotensin II in vascular smooth
muscle cells by phosphorylating the exchanger at serine/threonine residues located between amino
acids 671 and 714 (27).
Finally, it has been suggested that Krebs-Henseleit (KH), or more broadly, crystalloid,
perfused hearts are "hyperoxic" and thus more susceptible to oxidative stress because the
perfusate is commonly equilibrated with 95% O2. As is the case for respiration, the pertinent
factor is not PO2 per se, but rather the oxygen concentration in the solution at the site of the
reaction. At the level of cardiac myocytes in the KH perfused heart, there are at least two
arguments suggesting the cells are not "hyperoxic". First, there is no change in the extravascular
O2 transport pathway except perhaps for interstitial edema which would lengthen the pathway.
Second, although the actual concentration of intracellular O2 is elusive and not agreed upon, NMR
measurements of intracellular O2 in perfused and in situ myocardium indicate that O2 delivery to
heart cells in KH perfused hearts is not greater than in situ (26). Futhermore, when the critical
level of intracellular O2 is measured (25), KH perfused hearts are marginally closer to hypoxia
than blood perfused hearts (12). Nevertheless, to the extent that ischemia/reperfusion injury is
the result of oxidative stress to the capillary endothelium, the crystalloid perfused heart model
used here may be more susceptible.
In conclusion, the results of these studies are the first to provide strong evidence that
acute exposure to 1 nM 17ß-estradiol (E2 - 20 min or less) inhibits the response of NHE1 to
acidification. That is, acute E2 exposure decreases the rate of proton efflux after NH4 washout in
HEPES medium. Furthermore, acute E2 exposure diminishes accumulation of cytosolic H, Na,
and Ca in hearts from ovariectomized rats during ischemia. Limiting the accumulation of these
ions was associated with improved recovery of left ventricular developed pressure and decreased
release of LDH during reperfusion. Additionally, E2-dependent effects on all ions were
diminished by addition of 1µM LNAME. These results are consistent with the previously
untested hypothesis that an NO-dependent effect of E2 inhibits NHE1 to limit Na and thereby
Ca uptake during ischemia. However thermodynamic arguments also illustrate that the effect of
E2 on [Ca]i is greater than can be accounted for by the effect of limiting cytosolic Na
accumulation on sarcolemmal NCX and thus suggest E2 inhibits NCX kinetically.
Finally, we speculate that regardless of findings related to chronic effects of E2 (e.g. those
related to hormone replacement therapy) there may be a role for acute E2 treatment in therapies
aimed at limiting myocardial ischemia-induced injury, including those designed for use in organ
preservation or bypass surgery.
Research described in this article was supported by Philip Morris USA Inc and HL21179. NMR
spectrometer expense was funded in part by NIH RR02511 and NSF PCM-8417289.
APPENDIX: Calculation of reversal potential for NCX.
The NCX reversal potential is calculated as ENCX = 3ENa - 2ECa = 3RT/F ln ([Na]o/[Na]i) - RT/F
ln ([Ca]o/[Ca]i) where subscripts o and i correspond to extra- and intracellular, respectively. The
assumption is made that the intra- and extracellular volumes in the heart are equal and unchanging
during the experiment (e.g. during ischemia K loss = Na uptake) and that cell water = 2.5 L/Kg
dry wt. (3). This allows one to calculate [Na]i in mM from NMR measure Na (mEq/Kg dry wt).
Nai and [Ca]i are measured using NMR as described. [Ca]o is assumed constant at 1.8 mM
throughout the experiment. [Na]o = perfusate [Na] =141 mM at the beginning of the experiment,
and during ischemia the change in [Na]i is equal and opposite to the change in [Na]o. Using these
assumptions for end ischemia in OVX+E2 from Figs. 3 and 4 we find [Na]i = 30 mM, [Na]o =
121 mM, [Ca]i = 202 nM, and [Ca]o = 1.8 mM such that ENCX = -126 mV.
Figure 1. In perfused hearts from ovariectomized rats (OVX) acute 17ß-estradiol (E2 - 1 nM )
diminishes the proton efflux rate after normoxic acidification and the effect of E2 is diminished by
1 µM LNAME. Proton efflux rate after NH4Cl washout (mM/min) is plotted vs treatment. *
p<0.05 vs OVX+E2; number of experiments are given in parentheses. Please note, in contrast to
the ischemia protocol (results summarized in all other figures), 2-deoxy-D-glucose was not used
to inhibit glycolysis during the normoxic acidification protocol, the results of which are described
in this figure.
Figure 2. In perfused hearts from ovariectomized rats (OVX) acute 17ß-estradiol (E2 - 1 nM )
diminishes the fall in intracellular pH during ischemia and this effect is limited by LNAME (1
uM). Intracellular pH is plotted vs. time before, during, and after ischemia with E2 (triangles) or
without E2 (circles) and with LNAME (closed symbols) or without LNAME (open symbols). *
p<0.05 vs OVX control; † vs OVX+E2; § vs OVX+LNAME; number of experiments are given in
parentheses. LNAME±E2 are not significantly different.
Figure 3. In perfused hearts from ovariectomized rats (OVX) acute 17ß-estradiol (E2 - 1 nM )
diminishes the increase in intracellular Na during ischemia and this effect is limited by LNAME
(1 uM). Intracellular Na (mEq/kg dry wt) is plotted vs. time before, during, and after ischemia
with E2 (triangles) or without E2 (circles) and with LNAME (closed symbols) or without
LNAME (open symbols). * p<0.05 vs OVX control; † vs OVX+E2; § vs OVX+LNAME;
number of experiments are given in parentheses.
Figure 4. In perfused hearts from ovariectomized rats (OVX) acute 17ß-estradiol (E2 - 1 nM )
diminishes the increase in cytosolic [Ca] during ischemia and this effect is limited by LNAME (1
uM). Cytosolic [Ca] (nM) is plotted vs. time before, during, and after ischemia with E2
(triangles) or without E2 (circles) and with LNAME (closed symbols) or without LNAME (open
symbols). * p<0.05 vs OVX control; † vs OVX+E2; § vs OVX+LNAME; number of
experiments are given in parentheses.
Figure 5. In perfused hearts from ovariectomized rats (OVX) acute 17ß-estradiol (E2 - 1 nM )
improves recovery of left ventricular developed pressure (LVDP). LVDP (% of baseline) is
plotted vs. time during reperfusion with E2 (triangles) or without E2 (circles) and with LNAME
(closed symbols) or without LNAME (open symbols). * p<0.05 vs OVX control; number of
experiments are given in parentheses.
Figure 6. In perfused hearts from ovariectomized rats (OVX) acute 17ß-estradiol (E2 - 1 nM )
diminishes release of lactate dehydrogenase (LDH) during reperfusion. Total LDH released
during reperfusion (IU/gm dry wt) is plotted vs. treatment. * p<0.05 vs OVX+E2; number of
experiments are given in parentheses.
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Proton efflux (mM/min)
80 60 40200 -20
806040 200 -20
Intracellular Na (mEq/kg dry)
80 60 40200 -20
Cytosolic Ca (nM)
30 20 100 0
LVDP (% of baseline)
31 Download full-text
LDH Release (IU/gm dry wt)